The overall objective of this work is to develop methods that can provide mechanistic information about the ways in which the chemical functionality of a nearby surface affects nucleic acid base- pairing and hybridization, and to use these methods to understand how surface chemistry can be used to control DNA self-assembly. DNA-based biomedical nanotechnologies rely directly on Watson-Crick base-pairing to achieve molecular recognition capable of directing self-assembly for a variety of diagnostic and targeting applications. While base-pairing in the solution phase is relatively well-understood, in many DNA nanotechnologies, base-pairing/hybridization is intended to occur in the vicinity of one or more interfaces/surfaces that may promote competitive non- specific interactions with nucleic acids due to the particular vicinal surface chemistry. A better understanding of surface effects on hybridization will ultimately lead to improvements in a wide range of DNA-based technologies. In particular, it will enable the design and preparation of improved surface-modification strategies. We propose to develop advanced single-molecule tracking methods using total internal reflection fluorescence microscopy (TIRFM) with single-molecule resolution, where resonance energy transfer (RET) is used to obtain simultaneous conformational information. This approach can identify direct molecule-by-molecule correlations between conformation and dynamic interfacial events (e.g. adsorption/desorption, interfacial mobility, conformational fluctuations, hybridization), providing mechanistic understanding of how vicinal surface chemistry affects both specific and non-specific DNA interactions. These methods will be used to study DNA dynamics and base- pairing on model surfaces that represent the most important examples of competing non-covalent interactions. This two-year plan focuses on understanding the interfacial dynamic behavior of (1) oligonucleotides that can self-hybridize to form stem-loop secondary structures, and (2) complementary oligonucleotide pairs.